Alizarin-based chromogenic substrates, their uses and composition containing same

ABSTRACT

An alizarin-based chromogenic substrate, a composition containing the substrate, and its use to detect the presence of an enzymatic activity are disclosed. The substrate is particularly applicable in the field of biological diagnosis.

This application is a U.S. National Stage of International applicationPCT/FR00/03465, filed Dec. 11, 2000, and published on Jun. 14, 2001 inthe French Language.

This invention concerns the detection of hydrolytic enzymes, inparticular saccharidases, esterases and peptidases, by means of the useof effective chromogenic substrates.

For many years, special substrates have been used to determine whetherenzymatic activities typical of microorganisms are present or not.Through the use of specific substrates, it is possible—on the basis ofwhether a reaction takes place or not—to characterize the nature of agenus of microorgansms, or distinguish between different strains and/orspecies belonging to a given genus.

Synthetic enzyme substrates are made up of two different parts: thefirst part is specific to the enzyme activity being tested for and willhereafter be referred to as the target part; the second part acts as amarker and will hereafter be referred to as the marker part.

Such special substrates may be either fluorescent or chromogenic. Infact, the second marker part or the product of its reaction with one ormore other compounds becomes fluorescent or chromogenic when it is nolonger associated with the first target part (in this context, refer toPatent Application PCT/FR99/00781 filed on behalf of the applicant).

This invention concerns chromogenic substrates based on the Alizarins orAnthrarobins which, when incorporated into a substrate form usually havesome coloration. However, the color due to the marker part becomesaccentuated and/or altered following hydrolysis which leads to theseparation of said marker part from the target part of the substrate.Preferably, the color properties of the product generated are furtherenhanced by virtue of the presence of a developer factor (e.g. a metalsalt or high pH).

The capacity of the Alizarins to form colored chelation complexes withmetals was discovered in the Nineteenth Century. Beginning in 1826 whenAlizarins were first isolated from the plant Rubia tinctorum, theirproperties as dyes were exploited in tinting fabrics.

A complete synthetic pathway for the Alizarins was developed by Graebeand Liebermann in 1869. In the same year, W. H. Perkin extended thenumber of different Alizarins which could be synthesized by substitutingdifferent groups at positions R₃, R₄, R₅, R₆, R₇ and R₈ of theanthracene nucleus (shown below):

The Alizarins are not in themselves dyes but rather form insolublepigments with metal oxides. For example, when a sulfone group issubstituted in at position R₃, chelation with an Aluminum salt givesrise to a bright scarlet-red color whereas chelation with a chromiumsalt gives a Bordeaux-like shade of Red. Taking another example,3-Nitroalizarin and 4-Nitroalizarin give complexes of different colorsaccording to the metal salts with which they are chelated. Theusefulness of the Alizarins in the dying industry is largely due to thestability of the chelation complexes they form with metals, be they inthe form of soaps, acids or the hydroxides of alkali metals.

Among the Alizarin derivatives which are easy to synthesize, it can benoted that 3-Aminoalizarin and 4-Aminoalizarin can be generated from3-Nitroalizarin and 4-Nitroalizarin. 4-Aminoalizarin is representedbelow:

This compound is of particular interest because it gives a purple colorin the presence of aluminum. Moreover, it is the starting point for thesynthesis—using a Skraup reaction which is familiar to those skilled inthe art—of the quinoline Alizarins, one of which, Alizarin a-quinoline(which is green) is represented below:

Substitution and ring-closure reactions make it possible to generate awhole series of modified A lizarins with differing properties.

The prior art also shows that these compounds already have biologicaland biomedical applications. Because of the speed with whichHydroxyanthraquinones react in the presence of chelation complexes, theyhave found a preferred application in tests to detect whether metals arepresent or not in biological specimens.

The anthrarobins (also referred to as Deoxyalizarin orAnthracene-1,2,10-triol) which are produced by the reduction ofAlizarins, are already familiar to those skilled in the art. Reductionis mediated by the action of Zinc hydroxide, ammonia, acid tin chloride,etc. This compound's general formula is as follows:

However, up till now, no reference has been made to one particular areain which Alizarins, Anthraquinoid derivatives and Anthrarobins findapplications, namely in the detection of enzyme activity. Thisnecessarily involves the synthesis of substrates in which at least onetarget part is conjugated with the Alizarin molecule. These substrateshave the advantage that they fail to react in the presence of metal ionsas long as no hydrolysis reaction has occurred to separate the twodifferent parts. The fact that the chelation complexes formed areinsoluble results in a number of significant advantages:

-   -   enhanced sensitivity, even at a low concentration, meaning that        only a small amount of substrate needs to be used,    -   the color of the hydrolysis product can be readily adapted to        the requirements of the particular application by modifying the        composition of the reaction medium, notably the levels of        polyvalent cations (a definition of which will be given in the        special points at the end of this description) it contains        and/or its pH.    -   they are easy to synthesize and a only a small amount of        substrate is needed (by virtue of the great sensitivity        mentioned above), both of which factors reduce production costs,    -   the colored product diffuses inefficiently so that colonies are        easy to resolve and distinguish, and    -   growth is relatively uninhibited since such only trace amounts        of substrate are necessary (by virtue of the great sensitivity        mentioned above).

Alizarin derivatives have been synthesized, mainly bound to glycosides,using a fairly classic pathway referred to as the Koenigs-Knorr method(Koenigs, W. and Knorr, E., Ber., 34, 957, 1901). α-glycosides aresynthesized using a modified version of the Helferich method (HelferichB. et al., Ber., 66, 378 (1933) and Ber., 77, 194 (1944). Otherderivatives are bound to short-chain fatty acids and esters ofphosphoric or sulfuric acid.

Hitherto, the substrates used have been, e.g.5-Bromo-4-chloro-3-indolyl-β-D-galactoside which will be dealt withlater in a comparative analysis with one of the substrates according tothe invention, namely Alizarin-β-D-galactoside.

In accordance with this invention, the substrates according to theinvention are substantially more effective than those covered in theprior art. Thus, they detect a greater number of species and/or strainsof microorganism for any specific enzyme activity being assayed.

The substrates according to the invention are mentioned to varyingextents in other documents.

Thus, an article by Masawaki. Teruyuki et al., “Selective solventextraction of ruberythric acid from madder roots and subsequenthydrolysis with β-glucosidase”, J. Ferment. Bioeng. (1996), 81(6),567–569, concerns a process for the extraction of anthraquinones—to beused as dyes—from madder roots, based on the use of a selective solvent.One of the aims was to extract Alizarins from Anthraquinones bound tosugars; one such species was Alizarin-2-o-primeveroside. In order toachieve this, they hydrolyzed Alizarin-2-o-primeveroside usingβ-glucosidase.

Another article published by Van der Plas, Linus H. W. et al.“Anthraquinone glycosylation and hydrolysis in Morinda citrifolia cellsuspensions. Regulation and function”, J. Plant. Physiol., (1998),152(2/3), 235–241, proposes a biological explanation for the presence ofa sugar-the glycosylated Primeveroside—in plant cells (see page 240column 1, paragraph 3). According to this, it is generated by thehydrolysis of certain Anthraquinones.

A final article published by Mateiu, J et al. “Microbial glucosidationof dihydroxyanthraquinones. General properties of the glucosidationsystem”, Folia Microbiol. (Prague) (1974), 19(4), 307–316 concerns the“glucosidation” activity of the B96 mutant strain of Streptomycesaureofaciens.

However, these are only distantly related to the Applicant's invention.Although it is true that all three mention substrates based on Alizarin(or other anthraquinones), their diversity is restricted (with mentionof only a few compounds), and all are generated by biological pathways(substrates combining a Primeveroside and produced by plants in thefirst two articles; substrates combining various glucosides and producedby the bacterium Streptomyces griseus in the third). Moreover, thesesubstrates are not used to develop diagnostic tests based on thedetection of enzymes.

To this effect, this invention concerns a chromogenic substrate todetect the presence of an enzyme activity, with the following generalformula:

in which:

-   -   R₁ is a target part or H, and R₂ is a target part or H, with at        least one out of R₁ and R₂ being a target part,    -   R₃ is H, SO₃H, Cl, Br, F, I, NO₂, NH₂, NR₉R₁₀, or an Acylamino        Aminoaryl or Aminoacylamino group of the type NHCOX, with X        being an Alkyl, Aryl or Aralkyl group or an α-amino acid residue        such as Alanine,    -   R4 is H, SO₃H, Cl, Br, F, I, NO₂, NH₂, NR₉R₁₀, OH or an        Acylamino Aminoaryl or Aminoacylamino group of the type NHCOX,        with X being an Alkyl, Aryl or Aralkyl group or an α-amino acid        residue such as Alanine,    -   according to a modification, R₃ and R₄ form bonds with one        another to create a ring with at least five sides, and        preferably six sides.    -   R₅, R₆, R₇ and R₈ each consist of one of the following atoms or        groups of atoms: H, a halogen (particularly Cl or Br), OH, SO₃H,        or an Alkyl or Alkoxy group, and    -   R₉ a R₁₀ are independently a Methyl, Alkyl, Aryl, Aralkyl group,        or one (either R₉ or R₁₀) is a ring structure (Piperidine,        Pyrrolidine, Morpholine, etc.) with the other (either R₁₀ or R₉)        being a Hydrogen atom.

In a special case, the ketone groups of the central ring are reduced toform hydroxide groups in which at least one of the hydrogen atoms mightbe replaced by a Methyl, Alkyl, Aryl or Aralkyl group.

This invention also concerns a chromogenic substrate to detect thepresence of an enzyme activity, with the following general formula:

in which:

-   -   R₁ is a target part or H, and R₂ is a target part or H, with at        least one out of R₁ and R₂ being a target part,    -   R₃ is H, SO₃H, Cl, Br, F, I, NO₂, NH₂, Nr₉R₁₀, or an Acylamino        Aminoaryl or Aminoacylamino group of the type NHCOX, with X        being an Alkyl, Aryl or Aralkyl group or an α-amino acid residue        such as Alanine,    -   R4 is H, SO₃H, Cl, Br, F, I, NO₂, NH₂, NR₉R₁₀, OH or an        Acylamino Aminoaryl or Aminoacylamino group of the type NHCOX,        with X being an Alkyl, Aryl or Aralkyl group or an α-amino acid        residue such as Alanine,    -   according to a modification, R₃ and R₄ form bonds with one        another to create a ring with at least five sides, and        preferably six sides.    -   R₅, R₆, R₇ and R₈ each consist of one of the following atoms or        groups of atoms: H, a halogen (particularly Cl or Br), OH, SO₃H,        or an Alkyl or Alkoxy group, and    -   R₉ a R₁₀ are independently a Methyl, Alkyl, Aryl, Aralkyl group,        or one (either R₉ or R₁₀) is a ring structure (Piperidine,        Pyrrolidine, Morpholine, etc.) with the other (either R₁₀ or R₉)        being a Hydrogen atom.    -   R₁₁, consists of one of the following atoms or groups of atoms:        H, SO₃H, Cl, Br, F, I, NO₂, NH₂, NR₉R₁₀, or an Alkyl, Aryl,        Aralkyl, Acylamino Aminoaryl or Aminoacylamino group of the type        NHCOX, with X being an Alkyl, Aryl or Aralkyl group or an        α-amino acid residue, and    -   R₁₂ consists of H, or a Methyl, Alkyl, Aryl or Aralkyl group.

In a special case in which one of the above-mentioned substrates ishydrolyzed, the marker part consists of an Alizarin which is carryingtwo hydroxyl groups at positions 1 and 2. The name of this compound is1,2-Dihydroxyanthraquinone-β-D-galactoside and its general formula, whenit is conjugated with a target part consisting of galactose, is:

The product generated when this compound is hydrolyzed by aβ-galactosidase forms the following chelation complex in the presence ofan iron salt:

In all the examples given above, R₁ is preferably H and R₂ is preferablythe target part.

More precisely, the target part consists of one of the followingspecies:

-   -   a Glycoside, consisting of mono-, di- or poly-saccharide        sub-units, joined to the hydroxyl group through an α or β        linkage,    -   an α-amino acid or a peptide,    -   an organic acid, such as —O—CO—(CH₂)_(n)—CH₃, in which n has a        value of between 0 and 20, or    -   Sulfate, Phosphate, Pyrosulfate, Pyrophosphate or        Phosphodiester.

In a special case, R₃ and R₄ are such that, when joined to one anotherthrough a C₃N chain (which might or might not be substituted) with the Npreferably adjacent to R₃ or R₄, a six-sided ring is formed.

According to a first embodiment of at least one substrate as describedabove for the detection of an enzyme activity , the use consists in:

-   -   bringing at least one substrate, the target part of which        matches the activity of the enzyme being assayed, into contact        with a sample suspected of containing at least one microorganism        which expresses the enzyme activity in question, and at least        one type of cation, and    -   monitoring for the formation of an insoluble, colored chelation        complex.

Preferably, the substrate is introduced into the presence of at leastone type of cation which is appropriate for the marker part released bythe enzyme activity.

Still preferably, the type of cation which can be used to form insolublechelation complexes consists of. Fe²⁺, Al³⁺, Mn²⁺, Sn²⁺ or Cu²⁺.

If at least two substrates are being used, as described above, in orderto make it possible to detect at least two different enzyme activities,the use consists in:

-   -   bringing at least two substrates, the target parts of which        match the activities of the two (at least) enzymes being        assayed, into contact with a sample suspected of containing at        least one microorganism which expresses the enzyme activities in        question, plus at least one type of cation, and    -   monitoring for the development of at least two different colors        or a third color.

According to the last case, the substrates are introduced into thepresence of at least one type of cation, preferably a single type ofcation, which is appropriate for the marker parts released by the enzymeactivities.

In all the examples, the use of at least one substrate as describedabove to detect the presence of an enzyme activity, or this use inparallel with a use already described previously, consists in:

-   -   bringing at least one substrate, the target part of which        matches the activity of the enzyme being assayed, into contact        with a sample suspected of containing at least one microorganism        which expresses the enzyme activity in question, in a reaction        medium with an appropriate pH, and    -   monitoring for the development of at least one color.

Preferably, when at least two substrates are being used, only one singletype of cation should be used, this cation being appropriate for all themarker parts released by the enzyme activities.

According to a preferred embodiment, the above-mentioned uses include anintermediate step consisting in allowing the microorganism(s) to grow inor on a medium which has been supplemented with the substrates.

If it is glycosidase activity which is to be detected, the target partused could be (among other possibilities):

-   -   Glucose,    -   Galactose,    -   Mannose,    -   Xylose,    -   Glucuronic acid, or    -   N-acetylglucosamine.

If it is phosphatase activity which is to be detected, the target partused could be (among other possibilities) Phosphoric acid or asubstituted derivative thereof

If it is sulfatase activity which is to be detected, the target partused could be (among other possibilities) Sulfuric acid or a substitutedderivative thereof

If it is lipase, phospholipase or esterase activity which is to bedetected, the target part used could be (among other possibilities):

-   -   A fatty acid (saturated or non-saturated), or a substituted        derivative thereof,    -   Acetic acid, or a substituted derivative thereof,    -   Butyric acid, or a substituted derivative thereof,    -   Octanoic acid, or a substituted derivative thereof, or    -   An esterified phosphate group such as inositol-1-phosphate.

The invention also concerns a formulation for the detection of at leastone strain and/or species of microorganism which includes at least onesubstrate as described above plus a culture medium.

If the formulation contains at least two substrates, the reactionproducts formed should have different colors so that distinction can bemade between the different enzyme activities expressed by at least onestrain and/or species of microorganism.

Preferably, the form in which the formulation is presented is liquid,semi-solid or solid (e.g. a dry form ready for resuspension in anappropriate solution).

Still preferably, the substrate is at a concentration of between 10 and500 mg/l, preferably between 30 and 150 mg/l, and more preferably still,50 mg/l.

Therefore, this invention concerns a novel substrate which, at firstfaintly colored, takes on a more marked color in the presence of, on theone hand a microorganism or an enzyme, the presence of which it isdesired to detect, and on the other hand (in some cases) a cation. Theinvention also concerns the uses to which such a substrate can be put,and a formulation containing this substrate.

Having cations present is particularly useful, although not absolutelyneessary; in the latter case, the formulation used would preferably havean alkaline pH. It is also possible to combine both of these conditions,i.e. the presence of a cation plus alkaline pH.

When different cations are used, the colonies of the microorganismsbeing tested take on different colors. Cations such as Iron, Manganese,Tin and Aluminum are used at very low concentrations in order to preventor minimize the inhibition of growth caused by free ions in the testspecimen.

However, high concentrations of certain metal ions induce selectiveinhibition which may be exploited to select particular microbialspecies, thus constituting an additional advantage.

SUBSTRATE SYNTHESIS 1°) Synthesis of Alizarin-2-β-D-glucoside

According to Robertson et al., (J. Chem. Soc. (1930), 1136 et (1933),1167), Alizarin is specifically glycosylated at the hydroxyl group inposition 2, although this addition can be made at position 1.Neverthelesss, conjugation at position 2 is easier.

This substrate was prepared using a modified version of the methoddescribed by Robertson (1933). A mass of 6 g of Alizarin was resuspendedin 70 ml of acetone and mixed with 70 ml of Potassium hydroxide solution(0.28 mol/l) in order to form a salt. Added to this was a mixture of 40ml of a 1:1 mixture of Ether and Acetone containing 6.6 g ofAceto-bromo-glucose. The mixture was stirred for about 14 hours. Then, 7ml of Potassium hydroxide solution (1.25 mol/l) were added, followed by15 ml of Aceto-bromo-glucose (0.6 mol/l) in Acetone. The premixture wasstirred for a further ten hours. The Ether and the Acetone wereevaporated off at low pressure and the pH was adjusted to about 5.5using glacial Acetic acid. The mixture containing some unreactedAlizarin was filtered and then washed in water before being driedovernight at 50° C.

The resultant yellow solid was resuspended in 70 ml of glacial Aceticacid and ///shaken therein for 5 minutes. This cooled it down prior tofiltration after which it was washed in Acetic acid. The filtrate wasthen left for one hour and separated from any residual Alizarin. Thisprocedure was then repeated at 10° C. to yield a dark green filtrate.

The product was dried and dissolved in 200 ml of Dichloromethane beforethe addition of 2 ml of Triethylamine. An Aluminum oxide was premixedinto the solution until thin layer chromatography (TLC) of test samplesshowed that no Alizarin remained. The Aluminum oxide was removed and theremaining solution was evaporated with rotary motion, yielding a yellowsolid. This solid was recrystallized from hot Ethanol which contained afew drops of Acetic acid. This gave 2.02 g of AlizarinTetra-acetyl-glucoside.

A mass of 1.1 g of Alizarin Tetra-acetyl-glucoside was resuspended in 60ml of Ethanol before the addition of 30 ml of aqueous Sodium hydroxide(0.125 mol/l) to give a red solution. This mixture was kept at 65° C.for 10 minutes and then cooled to 0° C. The red glycosylated AlizarinSodium salt was then removed by vacuum filtration, washed with Ether andfinally dried. The procedure yielded 0.9 g of the Sodium salt ofAlizarin-2-β-D-glucoside. This product can be further purified usingtechniques familiar to those skilled in the art to yieldAlizarin-2-β-D-glucoside.

2°) Synthesis of Alizarin-2-β-D-galactoside

This substrate was also prepared using a modified version of the methoddescribed by Robertson (1933). A mass of 6 g of Alizarin was resuspendedin 70 ml of acetone and mixed with 70 ml of Potassium hydroxide solution(0.28 mol/l) in order to form a salt. Added to this was a mixture of 40ml of a 1:1 mixture of Ether and Acetone containing 6.6 g ofAceto-bromo-galactose. The mixture was stirred for about 14 hours. Then,7 ml of Potassium hydroxide solution (1.25 mol/l) were added, followedby 15 ml of Aceto-bromo-galactoside (0.6 mol/l) in Acetone. Thepremixture was stirred for a further ten hours. The Ether and theAcetone were evaporated off at low pressure and the pH was adjusted toabout 5.5 using glacial Acetic acid. The mixture containing someunreacted Alizarin was filtered and then washed in water before beingdried overnight at 50° C.

The product was dried and dissolved in 200 ml of Dichloromethane beforethe addition of 2 ml of Triethylamine. An Aluminum oxide was premixedinto the solution until thin layer chromatography (TLC) of test samplesshowed that no Alizarin remained. The Aluminum oxide was removed and theremaining solution was evaporated with rotary motion, yielding a yellowsolid. This solid was recrystallized from hot Ethanol which contained afew drops of Acetic acid. This gave 1.96 g of AlizarinTetra-acetyl-galactoside.

A mass of 1.1 g of Alizarin Tetra-acetyl-galactoside was resuspended in60 ml of Ethanol before the addition of 30 ml of aqueous Sodiumhydroxide (0.125 mol/l) to give a red solution. This mixture was kept at65° C. for 10 minutes and then cooled to 0° C. The red glycosylatedAlizarin Sodium salt was then removed by vacuum filtration, washed withEther and finally dried. The procedure yielded 0.86 g of the Sodium saltof Alizarin-2-β-D-galactoside in the form of a crystalline, red powder.

3°) Synthesis of Alizarin-2-acetate

This substrate was prepared using a method familiar to those skilled inthe art. Two grams of Alizarin were dissolved in 5 ml of Pyridine andtreated with a mixture of 2.5 ml of Acetic anhydride and 5 ml ofPyridine. After 16 hours at room temperature, the yellow solution waspoured into 100 ml of hydrochloric acid containing ice. The precipitatedAcetate was recovered by filtration with aspiration and then washed inwater. Recrystallization from Acetone yielded 1.4 g ofAlizarin-2-acetate in the form of yellow crystals.

4°) Synthesis of Alizarin-2-sulfate

This substrate was prepared by heating 2.4 g of Alizarin (i.e. 10 mmol)in 10 ml of pyridine containing 4 g of a Pyridine-Sulfur trioxidecomplex. After 2 hours at 60° C., the Pyridine was removed at lowpressure. The sulfate ester was recrystallized in the same way as thePotassium salt, by careful addition of Potassium hydroxide in methanoluntil a pH of 9 was reached. The Potassium salt forms gradually and wasrecovered by filtration with aspiration before being washed in Ether toyield 1.2 g of a white crystalline powder, Alizarin-2-sulfate.

5°) Synthesis of Alizarin-1-galactoside

This substrate was prepared from the Alizarin-2-acetate (the synthesisof which was described in Chapter 3°). A mass of 2,82 g of the ester(i.e. 10 mmol) was mixed into 75 ml of Dichloromethane and to thismixture was added 2 to 3 ml of either 2,4,6-Collidine or 2,6-Lutidine togenerate a deep purple solution. After one hour, silvercarbonate—prepared according to the method of Wolfrom and Lineback,“Methods in Carbohydrate Chemistry 2” (1963), 342–43—was added followedby 5 g (i.e. 12.5 mmol) of Aceto-bromo-galactose. The reaction isallowed to proceed at room temperature (i.e. 10 to 15° C.) for two daysin a constantly stirred vessel. Thin layer chromatography revealed thatsteady conversion to Tetra-acetyl-galactoside (which migrates fast onthe plate) was occurring. The solution was filtered through a Silica ora Diatomite bed and the filter aid was washed using sufficient (i.e.about 100 ml) Dichloromethane. The solution of the compound inDichloromethane was then washed in 0.2 M hydrochloric acid (3×100 ml) inwater (×2). After drying (MgSO₄), the light brown-colored extract wasevaporated at low pressure and then dissolved in a new aliquot ofmethanol. Thin layer chromatography (Ethyl acetate/Toluene) revealed thepresenbce of a fast-migrating species which gave a positive signal withultraviolet radiation and sulfuric acid. The protected galactoside wasthen deacetylated—using Sodium methoxide in Methanol as alreadydescribed—yielding 1.32 g of Alizarin-1-galactoside.

6°) Synthesis of Alizarin-1-phosphate-2-octanoate

This substrate was prepared by reacting 2.4 g (i.e. 10 mmol) in 100 mlof Dichloromethane and 3 ml of Triethylamine. To this solution, 1.62 gof Octanoyl chloride (10 mmol) was slowly added over a period of 30minutes at room temperature, stirring throughout. The Octanoate waspurified by treating it with Aluminum, as has already been described,and then recovered by removing the solvent and recrystallizing theproduct from Methanol. After cooling to −12° C., a mass of 1.84 g of theOctanoate (5 mmol) in 30 ml of dry Acetonitrile was successively treatedwith:

-   -   3.6 g of carbon tetrachloride,    -   1.6 g of Diisopropylethylamine, and    -   80 mg of 4-dimethylaminopyridine.        After about 2 minutes at low temperature, a solution containing        2.2 g of Dibenzyl phosphite was added in 8 ml of Acetonitrile        which prevents the temperature rising too high. After one hour,        the mixture was treated as described by Silverberg L. J.,        Dillon J. L. et Vermeshetti P., Tet. Lett., 37 N°6 (1996), and        the dibenzyl-phosphoryl ester was destroyed using Hydrogen by        using 30 ml of Ethyl acetate as solvent, in the presence of 0.4        g of a Palladium/Carbon catalyst (10% w/w). The Phosphate ester        was then recovered in the same way as the Potassium salt,        following removal of the acetate by mixing the solution with        Methanol and then carefully adding a solution of Potassium        carbonate in aqueous Methanol with a pH of 8. The precipitated        Potassium salt of Alizarin-1-phosphoric acid-2-octanoate was        recovered, washed in Methanol, and the 2.05 g of the Ester were        vacuum dried and used without any further purification.

7°) Synthesis of Deoxyalizarin

This type of substrate is prepared following the pathway given byLiebermann—Ber., 21 444 (1888). To synthesize 9- and 10-O-Methylderivatives of reduced Anthraquinones, all the 1,2-diol groups are firstprotected with methyl groups using the appropriate procedure (which isfamiliar to those skilled in the art) which consists in forming acomplex with borate, as described in Scheline—Acta. Chem. Scand. 20 1182(1966), or by using Acetaldehyde di-methyl acetate (protection byEthylidene).

APPLICATIONS

Many applications are possible:

-   1. Detection and identification of particular microbial species in    semi-solid media or on membranes.-   2. Detection of enzyme activities in solutions containing extracts    derived from tissues or cells, or in suspensions of eukaryotic or    prokaryotic cells.-   3. Identification of organisms on the basis of the enzyme activities    that they express.-   4. Visualization and localization of a specific reaction between an    antigen and an antibody, as in ELISA methods. For example,    Alizarin-β-galactoside or Alizarin-phosphate can be used for methods    designed to detect β-galactosidase or alkaline phosphatase    activities in assays for marker enzymes. In this case, the enzyme    substrates are first used in detection reactions in which an enzyme    activity (notably those of alkaline phosphatase or β-galactosidase)    is involved, as for reactions designed to detect antibodies or    antigens in an ELISA-type format, e.g. “Immunoassays: from Theory to    Practice” edited by Y. Barbier and published by ACOMEN, Lyon, pages    109 to 133 (1988); or otherwise, in the detection of nucleic acids,    e.g. “DNA probes” 2^(nd) Edition, Keller G. H., Manak, M. M.,    Stockton press, sections 5 to 9 (1993).-   1. Techniques of molecular biology designed to detect the presence    of a gene, e.g. that encoding β-galactosidase and its use “Molecular    cloning: a laboratory manual” 2^(nd) Edition, Sambrook, Fritsch,    Maniatis, Cold Spring Harbor Laboratory Press, sections 16.56 and    section 1.85 (1989).-   2. Techniques of histochemistry, cytochemistry and flow cytometry.    The uses of such enzyme-specific substrates which are important in    applications in medical diagnosis and other fields, e.g. water    quality testing, environmental testing, the food industry, etc.-   3. Detection of enzymes on polyacrylamide gels or other materials    used for electrophoresis and other separation methods.

EXAMPLES Example 1 Effect of Alizarin-2-β-D-galactoside Concentration onthe Detection of β-galactosidase Activity Due to Microorganisms in aSemi-solid Medium

A Columbia-base medium (46.37 g/l) was supplemented with eitherAlizarin-2-β-D-galactoside (0,01 g/l), 0,03 g/l, 0,05 g/l et 0,08 g/l)with Ammoniacal Iron Citrate (0,05 g/l), or with6-Chloro-3-indoyl-β-D-galactoside (0,2 g/l) without any Ammoniacal IronCitrate Petri dishes were prepared using these four different media (20ml of medium per dish). These dishes were divided into three areas andthen each area was inoculated with a bacterial suspension (density=0.5McFarland). The dishes were incubated for 48 hours at 37° C. Thecolonies which grew were examined by eye after 18, 24 and 48 hours ofincubation. Both the color and the intensity of the color were recorded.The results are presented in Table 1 below.

TABLE 1 Effect of substrate concentration on the detection ofβ-galactosidase activity due to microorganisms in a semi-solid medium6-Chloro-3-indolyl- β-D-galactoside Alizarine-2-β-D-galactosideAlizarin-2-β-D-galactoside Incubation 0.2 g/l 0.01 g/l 0.03 g/l 0.05 g/l0.08 g/l Strain time Color Intensity Color Intensity Color IntensityColor Intensity Color Intensity Escherichia coli 18 H pink 3.5 purple0.3 purple 2.7 purple 3.5 purple 4 115 24 H pink 3.5 purple 0.3 purple2.7 purple 3.5 purple 4 48 H pink 3.5 purple 0.5 purple 3 purple 3.5purple 4 Klebsiella pneumoniae 18 H — — — — purple 0.3 purple 0.8 purple1.5 023 24 H — — — — purple 0.3 purple 1 purple 1.5 48 H pink 0.3 — —purple 0.5 purple 1 purple 1.7 Enterococcus faecalis 18 H — — purple 0.3purple 2.7 purple 4 purple 4 117 24 H — — purple 0.6 purple 3 purple 4purple 4 48 H — — purple 0.6 purple 3.5 purple 4 purple 4 Serratiamarcescens 18 H pink 0.3 — — purple 0.5 purple 1 purple 2 042 24 H pink0.8 — — purple 1 purple 2.5 purple 2.5 48 H pink 3.5 purple 0.3 purple1.7 purple 3 purple 3.5 Proteus vulgaris 18 H — — — — — — — — — — 087 24H — — — — — — — — — — 48 H — — — — — — — — — — Morganella morganii 18 H— — — — — — — — — — 060 24 H — — — — — — — — — — 48 H — — — — — — — — ——

In Table 1, the sign “−” signifies no coloration. The strains which gavenothing but negative results are used as negative controls.

From Table 1, it can be seen that Alizarin-2-β-D-galactoside at aconcentration of 0.03 g/l gives an intensity of color very close to thatobserved with 6-Chloro-3-indolyl-β-D-galactoside at a concentrationwhich is about seven times higher. The range of concentrations at whichAlizarin-2-β-D-galactoside gives useful intensities of color is between0.03 g/l et 0.08 g/l, preferably 0.05 g/l. Alizarin-based substrates aretherefore more sensitive than Indoxyl-based ones.

Example 2 Detection of β-galactosidase Activity Due to Microorganisms ona Semi-solid Medium—Use of Alizarin-2-β-D-galactoside

Semi-solid medium supplemented with Alizarin-2-β-D-galactoside wasprepared as follows: 46.37 g of Columbia agar were added to 1 liter ofdistilled water together with 50 mg of Alizarin-2–13-D-galactoside, 500mg of Ammoniacal iron citrate and 30 mg of Isopropyl-β-D-thiogalactosideto induce β-galactosidase activity. The medium was sterilized byautoclaving at 116° C. for 10 minutes. It was then slowly cooled to 55°C. at which temperature it was poured into 20 ml Petri dishes. Thismedium was compared with another semi-solid medium prepared in the sameway using 46.37 g of Columbia agar together with 80 mg of5-Bromo-4-chloro-3-indolyl-β-D-galactoside and 30 mg ofIsopropyl-β-D-thiogalactoside.

Three-hundred-and-sixty-seven (367) different strains collected fromclinical and environmental specimens were identified using 20E API(Registered Trademark) strips (bioMérieux, France) as the referencemethod. All strains were grown on Columbia agar at 37° C. for 24 hoursand then an inoculum of about 10⁸ organisms/ml (equivalent to aMcFarland reading of 0,5) was prepared for every strain. Using a Denleyinoculator, one dish of each of the media was inoculated with 1microliter of each suspension, i.e. both the medium supplemented withAlizarin-2-β-D-galactoside prepared as described above, and the mediumcontaining 5-Bromo-4-chloro-3-indolyl-β-D-galactoside. All the disheswere incubated for 18 hours at 37° C.

After incubation, the colonies which had grown were inspected by eye. Onthe medium containing the Alizarin-2-β-D-galactoside, the coloniesexpressing β-galactosidase activity were purple in color whereas on themedium containing the 5-Bromo -4-chloro-3-indolyl-β-D-galactoside, theywere turquoise. Any strain showing either of these colors was consideredas being positive for β-galactosidase with the corresponding substrate.The results are presented in Table 2 below.

TABLE 2 Detection of β-galactosidase activity due to microorganisms on asemi-solid medium supplemented with Alizarin-2-β-D-galactoside5-Bromo-4-chloro- Number of strains Alizarin-2-β- 3-indolyl-2-β- Speciestested per species D-galactoside D-galactoside Acinetobacter spp. 53 0 0Aeromonas caviae 7 86 86 Aeromonas hydrophila 3 100 100 Citrobacterdiversus 9 89 89 Citrobacter freundii 16 100 100 Enterobacter aerogenes9 100 100 Enterobacter agglomerans 1 100 100 Enterobacter cloacae 21 100100 Escherichia coli 41 95 95 Escherichia hermannii 1 100 100 Hafniaalvei 10 80 80 Klebsiella oxytoca 13 100 100 Klebsiella ozaenae 3 100 67Klebsiella pneumoniae 19 100 100 Morganella morganii 12 0 0 Proteusmirabilis 16 0 0 Proteus penneri 1 0 0 Proteus vulgaris 4 0 0Providencia alcalifaciens 3 0 0 Providencia rettgeri 3 0 0 Providenciastuartii 10 0 0 Salmonella spp. 64 0 0 Serratia odorifera 1 100 100Serratia spp. 14 86 79 Shigella boydii 1 0 0 Shigella dysenteriae 2 0 0Shigella flexneri 2 0 0 Shigella sonnei 10 100 100 Vibrio cholerae 1 100100 Yersinia enterocolitica 14 64 29 Yersinia pseudotuberculosis 3 67 0

The figures given in the Alizarin-2-β-D-galactoside and5-Bromo-4-chloro-3-indolyl-β-D-galactoside columns correspond to thepercentage of positive strains. The vast majority of strains (96.5%)reacted in exactly the same way—i.e. either positively ornegatively—with both substrates. Eight strains selectively hydrolyzedAlizarin-2-β-D-galactoside. Nevertheless, all eight of these strainspossessed β-galactosidase activity as shown by the production of afluorescent signal in the presence of the substrate4-Methylumbelliferyl-β-D-galactoside. The results in this Tabletherefore show that the substrate is an effective marker forβ-galactosidase activity with a greater sensitivity than that observedwith the reference substrate,5-Bromo-4-chloro-3-indolyl-β-D-galactoside. Therefore, these substratesare extremely sensitive and can be used at very low concentrations.

Example 3 Effect of Using Different Metal Salts on the Color of theMarker Part

To the Columbia-base medium (46.37 g/l) supplemented withAlizarin-2-β-D-glucoside (50 mg/l), were added either Manganesechloride, Ammoniacal iron citrate, Tin chloride, or Aluminum sulfate,all at a concentration of 50 mg/l. A control medium with no metal saltswas tested in parallel. After autoclaving, these various media were allused to prepare Petri dishes (20 ml per plate). These dishes weredivided into three areas and then each area was inoculated with asuspension (density=0.5 McFarland) of microorganisms taken from theApplicant's collection. The dishes were incubated for 48 hours at 37° C.The colonies which grew were examined by eye after 24 and 48 hours ofincubation. Both the color and the intensity of the color were recorded.The results are presented in Table 3 below. It should be pointed outthat intensity readings are in arbitrary units. The only purpose ofgiving these values is to make it possible to compare one strain withanother in this respect. The same is true for the examples which follow.Similarly, the strains tested were taken from the Applicant's collectionand the number given represents an internal reference specific to thiscollection. The same internal numbering system is also used in some ofthe examples given later on.

TABLE 3 Effect of using different metal salts on the color of the markerpart Incubation Control Manganese chloride Iron citrate Tin chlorideAluminium sulfate Strain time Color Intensity Color Intensity ColorIntensity Color Intensity Color Intensity Listeria monocytogenes 24 Hpurple 2 purple 2.7 purple 4 orange 3 red 4 023 48 H purple 2 purple 2.7purple 4 orange 4 red 4 Listeria monocytogenes 24 H purple 2.3 purple2.7 purple 4 orange 3.5 red 4 079 48 H purple 2.3 purple 2.7 purple 4orange 4 red 4 Listeria monocytogenes 24 H purple 2.3 purple 2.3 purple4 orange 3 red 4 081 48 H purple 2.3 purple 2.7 purple 4 orange 4 red 4Listeria ivanovii 24 H purple 2.3 purple 2.7 purple 4 orange 3 red A 01848 H purple 2.3 purple 2.7 purple 4 orange 4 red 4 Listeria ivanovii 24H purple 2.3 purple 2.7 purple 4 orange 3 red 4 020 48 H purple 2.3purple 2.7 purple 4 orange 4 red 4 Listeria ivanovii 24 H purple 2.3purple 2.3 purple 4 orange 3 red 4 032 48 H purple 2.3 purple 2.3 purple4 orange 4 red 4 Listeria innocua 24 H purple 2.3 purple 2.3 purple 4orange 3 red 4 036 48 H purple 2.7 purple 2.7 purple 4 orange 4 red 4Listeria innocua 24 H purple 2 purple 2.3 purple 4 orange 3 red 4 029 48H purple 2.3 purple 2.3 purple 4 orange 4 red 4 Listeria innocua 24 Hpurple 2.3 purple 2.3 purple 4 orange 3 red 4 077 48 H purple 2.3 purple2.3 purple 4 orange 4 red 4 Listeria seeligeri 24 H purple 2.3 purple2.7 purple 4 orange 3 red 4 011 48 H purple 2.3 purple 2.7 purple 4orange 4 red 4 Listeria welshimeri 24 H purple 2.3 purple 2.3 purple 4orange 3 red 4 023 48 H purple 2.3 purple 2.7 purple 4 orange 4 red 4Listeria grayi 24 H purple 2 purple 2 purple 4 orange 3 red 4 078 48 Hpurple 2.3 purple 2 purple 4 orange 4 red 4

The color observed following hydrolysis of the substrate variesaccording to the metal salt used. Color can even be observed in theabsence of any metal salts (i.e. in the control medium). However, thecolor is less intense than that seen in the presence of a metal salt,e.g. the Iron salt (which does not affect the actual color).

This means that both the color and its intensity can be varied to matchspecific requirements (e.g. when used in combination with other enzymesubstrates or a pH indicator).

Example 4 Effect of pH on β-D-galactosidase Detection in the Presence ofAlizarin-2-β-D-galactoside

To each of eighteen tubes containing 5 ml of osmotically purified water,the following were added Alizarin-2-β-D-galactoside (0.05 g/l) and 5 μlof 62 -galactosidase (EC 3.2.1.23 Sigma). Ammoniacal iron citrate (0.05g/l) was added to half of these tubes. All 18 tubes were incubated for 4hours at 37° C., after which the tubes which did not contain anyAmmoniacal iron citrate had a light pink color, whereas the pinkcoloration in those which had been supplemented with Ammoniacal ironcitrate was observed to be more intense. Finally, the pH of these tubeswas adjusted to different values (2–3–4–5–6–7–8–9–10) at 24° C. Thecolors observed at the different PH readings are presented in Table 4below.

TABLE 4 Effect of pH on β-D-galactosidase detection in the presence of2- Alizarin-β-D-galactoside. pH Tubes containing no iron Tubessupplemented with iron pH 2 Yellow Yellow pH 3 Yellow Yellow pH 4 YellowYellow pH 5 Yellow - Orange Yellow - Pink pH 6 Pink - Orange Pink pH 7Pink - Orange Pink pH 8 Pink Purple pH 9 Purple Purple  pH 10 MauveMauve

The color changes with pH. As a general rule, at low pH it is yellow,and at higher pH it is pink or purple. This means that the color can bevaried to suit requirements or to detect different metabolic parameters(e.g. enzyme hydrolysis and pH variations). This experiment also showsthat a wide range of different pH values can be used.

Example 5 Combining an Alizarin-based Substrate with Another Substratein a Semi-solid Medium-detection of at Least Two Different EnzymeActivities

The following medium:

-   -   Columbia base (46.37 g/l),    -   Alizarin-2-β-D-glucoside (0,05 g/l),    -   Ammoniacal iron citrate (0.05 g/l),    -   5-Bromo-4-chloro-3-indolyl-β-D-galactoside (0,05 g/l), and    -   Isopropyl-β-D-thiogalactoside (30 mg/l) to induce        β-galactosidase activity, was used to pour Petri dishes (30 ml        per plate). These dishes were divided into three areas and then        each area was inoculated with a suspension (density=0.5        McFarland) of microorganisms taken from the Applicant's        collection. The dishes were incubated for 48 hours at 37° C.

The colonies which grew were examined by eye after 24 and 48 hours ofincubation. Both the color and the intensity of the color were recorded.The results are presented in Table 5 below.

TABLE 5 Simultaneous testing of two substrates: Alizarin-2-β-D-glucosideand 5-Bromo-4-chloro-3-indolyl-β-D-galactoside Alizarin-2-β-Glucosideplus Incubation 5-Bromo-4-chloro-3-indolyl-β-D-galactoside Strain timeColor Intensity Escherichia coli 24 H Turquoise 3.5 115 48 H Turquoise 4Escherichia coli 24 H Turquoise 3.5 206 48 H Turquoise 4 Citrobacterfreundii 24 H Turquoise 3.5 136 48 H Turquoise 3.5 Enterococcus faecalis24 H Purple 4 117 48 H Purple 4 Enterococcus faecalis 24 H Purple 4 06648 H Purple 4 Enterococcus faecium 24 H Purple 4 039 48 H Purple 4Klebsiella pneumoniae 24 H Blue Mauve 4 023 48 H Blue Mauve 4Enterobacter cloaecae 24 H Blue Mauve 4 059 48 H Blue Mauve 4Citrobacter koseri 24 H Blue Mauve 4 002 48 H Blue Mauve 4 Morganellamorganii 24 H — — 035 48 H — — Pseudomonas aeruginosa 24 H — — 054 48 H— — Proteu mirabilis 248 H  — — 154 48 H — —

In Table 5, the sign “−” signifies no coloration. The strains which gavenothing but negative results are used as negative controls.

By using a combination of two different substrates, four differentgroups of microorganism can be distinguished:

-   -   the first group, members of which give a turquoise color,        corresponds to species which express only β-galactosidase        activity,    -   the second group, members of which give a purple color,        corresponds to species which express only β-glucosidase        activity,    -   members of the third group show a hybrid of the two patterns        above, namely a blue or mauve color, and correspond to species        which express both of the enzyme activities being assayed, and    -   the fourth, colorless group corresponds to species which express        neither of the above-mentioned activities.

It is therefore possible, using Alizarin-based substrates combined withother enzyme substrates, to distinguish between one or more groups ofmicro-organisms on the basis of the biochemical activities that theyexpress.

Example 6 Detection of β-glucosidase Activity Due to Microorganisms on aSemi-solid Medium—Use of Alizarin-2-β-D-glucoside

Into a well of an API (Registered trademark, bioMérieux, France) strip,3 μl of a mixture of Alizarin-2-β-D-glucoside (0.12 g/l) and Ammoniacaliron citrate (0.05 g/l) were introduced and then dried. A control wellcontaining a final concentration of 0.4 g/l of6-Chloro-3-indolyl-β-D-glucoside was set up in parallel. Then 50 μl ofColumbia base were added to both of these wells which were subsequentlyinoculated with 100 μl of a bacterial suspension (density=2 McFarland).After 4 and 24 hours of incubation at 37° C., the color generated in thewells was recorded. The results are presented in Table 6 below.

TABLE 6 Detection of β-glucosidase activity due to microorganisms on asemi-solid medium supplemented with Alizarin-2-β-D-glucoside IncubationColor observed with the Color observed with the 6- Strain timeAlizarin-2-β-D-glucoside Chloro-3-indolyl-β-D-glucoside Listeriamonocytogenes  4 H Purple Pink 022 24 H Purple Pink Listeria ivanovit  4H Purple Pink 018 24 H Purple Pink Listeria innocua  4 H Purple Pink 03624 H Purple Pink Listeria seeligeri  4 H Purple — 080 24 H Purple PinkBacillus thuringiensis  4 H — — 072 24 H Purple — Klebsiella pneumoniae 4 H Purple Pink 023 24 H Purple Pink Staphylococcus aureus  4 H — — 06224 H — — Enterococcus faecium  4 H Purple Pink 009 24 H Purple Pink

In Table 6, the sign “−” signifies no coloration. The strains which gavenothing but negative results are used as negative controls.

The Alizarin-based substrate detected activity in. seven (7) of theeight (8) species tested, whereas 6-Chloro-3-indolyl-β-D-glucoside onlydetected such activity in 6) of these species. Moreover, in the case oftwo of these species (Bacillus thuringiensis and Listeria seeligeri),their activity manifested at an earlier time point with theAlizarin-based substrate. Therefore, these substrates can not only beused in liquid broth, but they are also more sensitive thanIndoxyl-based substrates.

Example 7 Comparison of Alizarin-1-β-D-glucoside andAlizarin-2-β-D-glucoside in Semi-solid Medium

To Columbia base, were added, either:

-   -   Alizarin-2-β-D-glucoside at 0.05 g/l plus Ammoniacal iron        citrate at 0.05 g/l,    -   Alizarin-1-β-D-glucoside at 0.05 g/l and 0.1 g/l plus, for both        concentrations, Ammoniacal iron citrate at 0.05 g/l,    -   6-Chloro-3-indolyl-β-D-glucoside at 0.15 g/l without any        Ammoniacal iron citrate.

Petri dishes were prepared using these four media (20 ml of medium perdish). These dishes were divided into three areas and then each area wasinoculated with a bacterial suspension (density=0.5 McFarland). Thedishes were incubated for 48 hours at 37° C. The colonies which grewwere examined by eye after 18, 24 and 48 hours of incubation. Both thecolor and the intensity of the color of the colonies were recorded. Theresults are presented in Table 7 below.

TABLE 7 Comparison of Alizarin-1-β-D-glucoside andAlizarin-2-β-D-glucoside in semi-solid medium. 6-Chloro-3-indolyl-β-D-1-Alizarin1-β-D-glucoside 1-Alizarin1-β-D-glucoside Incubation glucoside2-Alizarin-β-D-glucoside (0.05 g/l), (0.1 g/l), Strain time ColorIntensity Color Intensity Color Intensity Color Intensity Escherichiacoli 18 H — — — — — — — — 115 24 H — — — — — — — — 48 H — — — — — — — —Citrobacter koseri 18 H Pink 0.5 Mauve 2 Mauve 1.5 Mauve 2.5 059 24 HPink 1 Mauve 3 Mauve 2 Mauve 3 48 H Pink 2 Mauve 3.5 Mauve 2.5 Mauve 4Enterococcus faecalis 18 H Pink 1.5 Purple 3.5 Purple 3.5 Purple 3.5 11724 H Pink 2 Purple 4 Purple 4 Purple 4 48 H Pink 2.5 Purple 4 Purple 4Purple 4 Klebsiella 18 H Pink 2 Purple 3.5 Purple 2.5 Purple 3.5pneumoniae 24 H Pink 2.5 Purple 4 Purple 3 Purple 4 023 48 H Pink 3Purple 4 Purple 3.5 Purple 4 Proteus mirabilis 18 H — — — — — — — — 00824 H — — — — — — — — 48 H — — — — — — — — Staphylococcus 18 H — — — — —— — — epidermidis 24 H — — — — — — — — 024 48 H — — — — — — — —

In Table 7, the sign “−” signifies no coloration. The strains which gavenothing but negative results are used as negative controls.

At an equivalent concentration of 0.05 g/l, the two substrates did notgive the same intensity of color with two (2) of the three (3) strainswhich expressed the activity. The Alizarin-2-β-D-glucoside is slightlymore sensitive than the Alizarin-1-β-D-glucoside. However, at aconcentration of 0.1 g/l, Alizarin-1-β-D-glucoside gave deeperintensities than Alizarin-2-β-D-glucoside.

From this Table, it can be seen that the intensities observed withAlizarin-1-β-D-glucoside at a concentration of 0.05 g/l were-for allstrains tested-deeper than those obtained with6-Chloro-3-indolyl-β-D-glucoside which was at a three-fold higherconcentration.

Alizarin-based substrates are therefore more sensitive thanIndoxyl-based ones.

The choice of whether to use Alizarin-1-β-D-glucoside orAlizarin-2-β-D-glucoside might be affected by other factors, such asproduction cost, stability, particulars of the application, etc.

SPECIAL POINTS

One method of visualizing and therefore identifying bacterial species incolonies involves growing them on a semi-solid medium supplemented withat least one substrate as synthesized above in the presence of tracequantities of at least one appropriate cation. The growing colonies thustake on an intense coloration (red, purple, blue, etc.) which depends onthe substrate used, and the cation associated with it. When a ratioexists between the substrate and the cation, it is between 1/100 and100/1, preferably between 1/10 and 10/1, and more preferably still,between 1/2 and 2/1.

In certain conditions, the presence of a SO₃H group at position R₃ ofthe substrate helps overcome problems of stability and solubilityassociated with this type of substrate.

The term “polyvalent cations” refers to multivalent metal cations of theX^(n+) type, with n=2, 3 or 4. The metals which can be used are: Mg, Al,Ca, Ti, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, Sr, Zr, Sn, Sb, Ba, La and Hf, aswell as the Lanthanides Ce, Sm, Eu, Gd and Tb.

The Anthrarobins (also referred to as Deoxyalizarin orAnthracene-1,2,10-triol) which are produced by the reduction ofAlizarin, are already familiar to those skilled in the art. Reduction ismediated by the action of Zinc hydroxide, ammonia, Acid tin chloride,etc. This compound's general formula is as follows:

Because of the absence of the central quinonoid ring of Anthraquinone,the reactivity of metal chelation complexes of the 1,2-diol system isdifferent, e.g. the ferrous chelation complex is black in color. SinceAlizarin ferrous chelation complexes are red, two different types ofsubstrate—to detect two different enzyme activities—can be used with thesame metal ion. This pair of substrates could be, for example:

-   -   Alizarin-2-β-D-glucoside to detect saccharidase activity, and    -   Deoxyalizarin-2-β-D-phosphatase to detect phosphatase activity.

It is also possible to protect the hydroxyl group at position 10 of theAnthrarobins in which—for example—the hydrogen atom can be substitutedby a methyl group giving a compound with the following structure:

Similarly, the Alizarins can alsobe protected by reducing the ketonegroups to generate a 9,10-diol structure; the alcohol groups can then bealkylated, e.g. to give 1,2-Dihydroxy-9,10-dimethoxyvanthracene, asrepresented in the following:

These alkylated Anthrarobins and Alizarins are not susceptible tospontaneous oxidization to form Alizarins and are, as a result,excellent candidates for the glycosidation of substrates, preferentiallyat position 2.

1. A method for detecting enzyme activity comprising bringing at leastone substrate, the target part of which matches the activity of theenzyme being assayed, into contact with a sample suspected of containingat least one microorganism which expresses said enzyme activity, and atleast one type of cation, and monitoring for the formation of aninsoluble, colored chelation complex, wherein said substrate hasfollowing general formula:

in which: R₁ is a target part or H, and R₂ is a target part or H, withat least one of R₁ and R₂ being a target part, R₃ is H, SO₃H, Cl, Br, F,I, NO₂, NH₂, NR₉R₁₀, or an acylamino, aminoaryl, or aminoacylamino groupof the type NHCOX, with X being an alkyl, aryl or aralkyl group or ana-amino acid residue, R₄ is H, SO₃H, Cl, Br, F, I, NO₂, NH₂, NR₉R₁₀, OHor an acylamino aminoaryl or aminoacylamino group of the type NHCOX,with X being an alkyl, aryl or aralkyl group or an a-amino acid residue,alternatively, R₃ and R₄ form bonds with one another to create a ringwith at least five sides, R₅, R₆, R₇ and R₈ are each independentlyselected from the group consisting of H, a halogen, OH, SO₃H, an alkylgroup and an alkoxy group, and R₉ and R₁₀ are independently an alkyl,aryl, aralkyl group, or either R₉ or R₁₀ is a ring structure with theother, either R₁₀ or R₉, being a hydrogen atom.
 2. The method of claim1, wherein the ketone groups of the central ring are reduced to formhydroxide groups in which at least one of the hydrogen atoms areoptionally replaced by an alkyl group, aryl group or aralkyl group.
 3. Amethod for detecting enzyme activity comprising Bringing at least onesubstrate, the target part of which matches the activity of the enzymebeing assayed, into contact with a sample suspected of containing atleast one microorganism which expresses said enzyme activity, and atleast one type of cation, and monitoring for the formation of aninsoluble, colored chelation complex, wherein said substrate has thefollowing general formula:

in which: R₁ is a target part or H, and R₂ is a target part or H, withat least one of R₁ and R₂ being a target part, R₃ is H, SO₃H, Cl, Br, F,I, NO₂, NH₂, NR₉R₁₀, or an acylamino aminoaryl or aminoacylamino groupof the type NHCOX, with X being an alkyl, aryl or aralkyl group or ana-amino acid residue, R₄ is H, SO₃H, Cl, Br, F, I, NO₂, NH₂, NR₉R₁₀, OHor an acylamino aminoaryl or aminoacylamino group of the type NHCOX,with X being an alkyl, aryl or aralkyl group or an a-amino acid residue,alternatively, R₃ and R₄ form bonds with one another to create a ringwith at least five sides, R₅, R₆, R₇ and R₈ are each independentlyselected from the group consisting of H, a halogen, OH, SO₃H, an alkylgroup and an alkoxy group, and R₉ and R₁₀ are independently an alkyl,aryl, aralkyl group, or one, either R₉ or R₁₀ is a ring structure withthe other, either R₁₀ or R₉, being a hydrogen atom, R₁₁ is a memberselected from the group consisting of H, SO₃H, Cl, Br, F, I, NO₂, NH₂,NR₉R₁₀, or an alkyl, aryl, aralkyl, acylamino aminoaryl oraminoacylamino group of the type NHCOX, with X being an alkyl, aryl oraralkyl group, and an a-amino acid residue, and R₁₂ is a member selectedfrom the group consisting of H, an alkyl group, an aryl group and anaralkyl group.
 4. The method of claim 1, wherein R₁ is H and R₂ is atarget part.
 5. The method of claim 1, wherein the target part is amember of the group consisting of: a glycoside, consisting of mono-, di-or poly-saccharide sub-units, joined to the hydroxyl group through” α orβ carbon, an “amino acid or a peptide”, an organic acid having a formula—O—CO—(CH₂)_(n)—CH₃, in which n has a value of between 0 and 20, andsulfate, phosphate, pyrosulfate, pyrophosphate or phosphodiester.
 6. Themethod of claim 1, wherein R₃ and R₄ are such that, when joined to oneanother through a substituted or unsubstituted C₃N chain, a six-sidedring is formed.
 7. The method of claim 1, wherein the substrate isintroduced in the presence of at least one type of cation, wherein saidcation is reactive for the colored chelation complex that is formed. 8.The method of claim 7, wherein said type of cation is a member selectedfrom the group consisting of Fe²⁺, Al³⁺, Mn²⁺, Sn²⁺ and Cu²⁺.
 9. Themethod of claim 1, wherein at least two substrates are used to detectthe presence of at least two different enzyme activities, wherein saidmethod comprises the following steps: bringing at least two substrates,the target parts of which match the activities of said at least twoenzymes being assayed, into contact with a sample suspected ofcontaining at least one microorganism which expresses the enzymeactivities of the two enzymes, plus at least one type of cation, andmonitoring for the development of at least two different colors or athird color.
 10. The method of claim 9, wherein the substrate isintroduced in the presence of at least one type of cation, wherein saidcation is reactive for all the chelation complexes that are formed. 11.The method of claim 1, said method comprising: bringing at least onesubstrate, the target part of which matches the activity of the enzymebeing assayed, into contact with a sample suspected of containing atleast one microorganism which expresses said enzyme activity, in areaction medium with an appropriate pH, and monitoring for thedevelopment of at least one color.
 12. The method of claim 1, wherein asingle type of cation is reactive for all the chelation complexes thatare formed.
 13. The method of claim 1, further comprising growing themicroorganism(s) on a culture medium supplemented with the substrate(s).14. The method of claim 1, wherein glycosidase enzyme activity isdetected, and the target part is selected from the group consisting ofglucose, galactose, mannose, xylose, glucuronic acid andN-acetylglucosamine.